Despite improvement in insulin delivery, maintaining tight control of glucose homeostasis continues to be a challenge that results in bouts of severe hypo and hyperglycemia and serious long term complications in many type 1 diabetes (T1D) patients. Therefore, developing an immunotherapy for the disease remains a major goal. Reaching this goal, however, requires deep knowledge of all facets of the diabetogenic process - which is generally believed to be initiated by an imbalance between pathogenic and regulatory mechanisms that allows diabetogenic T cells to infiltrate pancreatic islets and destroy insulin-producing ss-cells. Therefore, identifying and understanding the roles of various molecules and cell types that tip the balance towards the immunopathogenic pathways in susceptible individuals and animal models is important for developing effective immunotherapy. This proposal investigates mechanisms that powerfully control ss-cell specific autoreactive T-cells when Fas ligand (FasL), an apoptosis-inducing member of tumor necrosis factor (TNF) family, is genetically or pharmacologically inactivated. Previously, the lack of appropriate models and efficacious FasL blocking monoclonal antibodies (mAb) has severely hampered such investigation. In this application, we will use NOD mice that are haploinsufficient for FasL (NOD-gld/+ mouse) and a FasL-neutralizing mAb (MFL4 clone) to investigate the underlying mechanisms and therapeutic significance of FasL blockade using the MFL4 mAb. NOD-gld/+ mice are completely protected from T1D, immunocompetent, and have normal immune homeostasis. In addition, MFL4 mAb protects NOD-wt mice from diabetes without altering immune homeostasis and more importantly our preliminary data show it has promising efficacy in reversing hyperglycemia in new-onset cases. Based on our preliminary data generated using these model systems, we hypothesize that an IL-10-producing CD5+ regulatory B cell subset that suppresses diabetogenic autoreactive T-cells are negatively regulated by FasL. In NOD mice, haploinsufficiency for FasL (gld/+) or mAb blockade of FasL prevents CD5+ regulatory B cell elimination, leading to control of diabetogenic T cells and suppression of insulitis (tested i Aim 1). In NOD-wt mice, FasL-mediated apoptosis eliminates CD5+ regulatory B cells thereby removing the brakes on autoreactive T-cells (tested in Aim 2). In addition, we hypothesize that the MFL4 mAb can be used to prevent disease in the late stages of insulitis and to reverse new-onset diabetes. Because the role of FasL in normal immune response and ss-cell death are dispensable, understanding how FasL modulates the diabetogenic process will lead to new mechanistic insights into the disease pathogenesis that could have important therapeutic implications.